| 1. |
Kang MH,
Ni H,
Jeffries TW,
( 2003 ) Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae. PMID : 12721450 : Abstract >>
Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.
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2. |
Schüte H,
Flossdorf J,
Sahm H,
Kula MR,
( 1976 ) Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. PMID : 1248477 : DOI : 10.1111/j.1432-1033.1976.tb10108.x Abstract >>
Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.
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3. |
Komeda T,
Sakai Y,
Kato N,
Kondo K,
( 2002 ) Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI). PMID : 12005059 : DOI : 10.1271/bbb.66.628 Abstract >>
The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.
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4. |
Horiguchi H,
Yurimoto H,
Goh T,
Nakagawa T,
Kato N,
Sakai Y,
( 2001 ) Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance. PMID : 11591682 : DOI : 10.1128/JB.183.21.6372-6383.2001 PMC : PMC100133 Abstract >>
In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.
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5. |
Stewart MQ,
Esposito RD,
Gowani J,
Goodman JM,
( 2001 ) Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments. PMID : 11683419 : Abstract >>
Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15 degrees C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.
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6. |
Horiguchi H,
Yurimoto H,
Kato N,
Sakai Y,
( 2001 ) Antioxidant system within yeast peroxisome. Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii. PMID : 11278957 : DOI : 10.1074/jbc.M011661200 Abstract >>
Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.
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7. |
Labrou NE,
Rigden DJ,
( 2001 ) Active-site characterization of Candida boidinii formate dehydrogenase. PMID : 11171126 : DOI : 10.1042/0264-6021:3540455 PMC : PMC1221675 Abstract >>
NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69-Ala, Asn-119-His, Ile-175-Ala, Gln-197-Leu, Arg-258-Ala, Gln-287-Glu and His-311-Gln) and double amino acid substitutions (Asn-119-His/His-311-Gln, Gln-287-Glu/His-311-Gln and Gln-287-Glu/Pro-288-Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311-Gln and Gln-287-Glu/His-311-Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate.
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8. |
Hagishita T,
Yurimoto H,
Nishikawa M,
( 2000 ) Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii. PMID : 10913603 : DOI : 10.1016/s0014-5793(00)01708-7 Abstract >>
Acetylspermidine oxidase (ASOD) belongs to a family of FAD-containing amine oxidases and catalyzes the oxidation of N-acetylated spermidine in polyamine metabolism. ASOD was purified to apparent homogeneity from cells of the methylotrophic yeast Candida boidinii grown on spermidine as the sole nitrogen source. C. boidinii ASOD catalyzed the oxidation of only N(1)-acetylspermidine. Based on partial amino acid sequences, oligonucleotide primers were designed for polymerase chain reaction, and the ASOD-encoding gene, ASO1, was cloned. The open reading frame encoding ASO1 was 1530 bp long and corresponded to a protein of 509 amino acid residues (calculated molecular mass=57167 Da). ASO1 contained a FAD-binding motif of G-A-G-I-A-G in the N-terminal region and carried an amino acid sequence of -S-K-L at the C-terminal, representing a typical peroxisome targeting signal 1. ASOD was localized in the peroxisomes in overexpressed C. boidinii. To our knowledge, this is the first report on the gene coding for ASOD that can catalyze the oxidation of N-acetylated polyamine as a substrate, from any type of organism.
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9. |
Suda H,
Sakai Y,
Komeda T,
( 2000 ) Cloning and sequence analysis of the Candida boidinii ADE2 gene. PMID : 10870106 : DOI : 10.1002/1097-0061(200007)16:10<953::AID-YEA590>3.0.CO;2-D Abstract >>
Candida boidinii ADE2 gene (phosphoribosyl-5-aminoimidazole carboxylase; AIRC, EC 4. 1. 1. 21) has been cloned by homology to the Saccharomyces cerevisiae ADE2 gene. The cloned C. boidinii ADE2 gene complemented the ade2 mutation of S. cerevisiae. Sequence analysis showed a single open reading frame of 1719 nucleotides coding for a polypeptide of 573 residues. Comparison of the deduced amino acid sequence with those of AIRC enzymes from other yeasts showed marked homology among yeast AIRCs.
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10. |
Labrou NE,
Rigden DJ,
Clonis YD,
( 2000 ) Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis. PMID : 11054119 : DOI : 10.1046/j.1432-1327.2000.01761.x Abstract >>
The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.
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11. |
Yurimoto H,
Hasegawa T,
Sakai Y,
Kato N,
( 2000 ) Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii. PMID : 10992285 : DOI : 10.1002/1097-0061(20000930)16:13<1217::AID-YEA616>3.0.CO;2-2 Abstract >>
A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.
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12. |
Felber S,
Slusarczyk H,
( 2000 ) Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues. PMID : 10691964 : DOI : 10.1046/j.1432-1327.2000.01123.x Abstract >>
The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.
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13. |
Schirwitz K,
Schmidt A,
Lamzin VS,
( 2007 ) High-resolution structures of formate dehydrogenase from Candida boidinii. PMID : 17525463 : DOI : 10.1110/ps.062741707 PMC : PMC2206666 Abstract >>
The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD(+)-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies.
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14. |
Cirino PC,
Chin JW,
Ingram LO,
( 2006 ) Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. PMID : 16838379 : DOI : 10.1002/bit.21082 Abstract >>
The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (approximately 275 mM in LB cultures, approximately 180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, DeltaxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of approximately 250 mM xylitol (38 g/L), with concomitant utilization of approximately 150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to > 3.7:1 using metabolically active "resting" cells.
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15. |
Sakai Y,
Tani Y,
( 1992 ) Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2. PMID : 1587486 : DOI : 10.1016/0378-1119(92)90708-w Abstract >>
Alcohol oxidase (AOD) is the first key enzyme for methanol metabolism in methylotrophic yeasts. AOD activity is strictly regulated by carbon source. The AOD1 gene was cloned from a gene library of the asporogenous formaldehyde-producing methylotrophic yeast, Candida boidinii S2. The complete nucleotide sequence of the gene and its 5'- and 3'-flanking regions (4174 bp) were determined. To identify the conserved and divergent sequences of the AOD1 gene and its 5'-flanking sequences among different species of methylotrophic yeasts, the AOD-encoding genes from C. boidinii S2 (AOD1), Hansenula polymorpha (MOX) and Pichia pastoris (AOX1 and AOX2) were compared. In addition to conserved amino acid sequences, several DNA segments in the G+C-rich region of 5'-flanking sequences were also found to be conserved. Northern analysis showed that the AOD1 gene transcript was induced by methanol, but was not detected when cells were grown on ethanol or glucose. Thus, as in ascosporogenous methylotrophic yeasts, AOD1 gene expression in C. boidinii appears to be controlled at the RNA level.
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16. |
Szamecz B,
Urbán G,
Rubiera R,
Kucsera J,
Dorgai L,
( 2005 ) Identification of four alcohol oxidases from methylotrophic yeasts. PMID : 16032762 : DOI : 10.1002/yea.1236 Abstract >>
Three yeast strains capable of utilizing methanol as sole carbon and energy source were isolated. Two were classified as Candida boidinii, while the third belonged in the genus Pichia. From these three strains, four alcohol oxidases genes were identified and the sequences of the coding regions were determined: one from each Candida boidinii (aox0673 and aox0680) and two from Pichia sp. 159 (aoxA and aoxB). Methanol induces both alcohol oxidases in Pichia sp. 159: the levels of aoxA and aoxB mRNA reach about 100% and 300%, respectively, of that of his4 mRNA. aoxA, but not aoxB, is expressed at a low level in the presence of glucose. The newly described alcohol oxidases have proper dinucleotide binding sites and PTS1-like C-terminal tripeptides, identified as important elements for peroxisomal localization.
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17. |
Sakai Y,
Tani Y,
( 1992 ) Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains. PMID : 1522074 : DOI : 10.1128/jb.174.18.5988-5993.1992 PMC : PMC207139 Abstract >>
A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.
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18. |
Yurimoto H,
Lee B,
Yasuda F,
Sakai Y,
Kato N,
( 2004 ) Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii. PMID : 15042594 : DOI : 10.1002/yea.1101 Abstract >>
Methyl formate synthesis during growth on methanol by methylotrophic yeasts has been considered to play a role in formaldehyde detoxification. An enzyme that catalyses methyl formate synthesis was purified from methylotrophic yeasts, and was suggested to belong to a family of alcohol dehydrogenases (ADHs). In this study we report the gene cloning and gene disruption analysis of three ADH-encoding genes in the methylotrophic yeast Candida boidinii (CbADH1, CbADH2 and CbADH3) in order to clarify the physiological role of methyl formate synthesis. From the primary structures of these three genes, CbAdh1 was shown to be cytosolic and CbAdh2 and CbAdh3 were mitochondrial enzymes. Gene products of CbADH1, CbADH2 and CbADH3 expressed in Escherichia coli showed both ADH- and methyl formate-synthesizing activities. The results of gene-disruption analyses suggested that methyl formate synthesis was mainly catalysed by a cytosolic ADH (CbAdh1), and this enzyme contributed to formaldehyde detoxification through glutathione-independent formaldehyde oxidation during growth on methanol by methylotrophic yeasts.
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19. |
Kurtzman CP,
Robnett CJ,
( 2010 ) Systematics of methanol assimilating yeasts and neighboring taxa from multigene sequence analysis and the proposal of Peterozyma gen. nov., a new member of the Saccharomycetales. PMID : 20522116 : DOI : 10.1111/j.1567-1364.2010.00625.x Abstract >>
The relatedness among methanol-assimilating yeasts assigned to the genus Ogataea and neighboring taxa (Phylum Ascomycota, Subphylum Saccharomycotina, Class Saccharomycetes, Order Saccharomycetales) was determined from phylogenetic analyses of gene sequences for nuclear large and small subunit (SSU) rRNAs, translation elongation factor-1alpha and mitochondrial SSU rRNA. On the basis of the analyses, Williopsis salicorniae and seven species of Pichia are proposed for transfer to the genus Ogataea, which has been emended, and Pichia angophorae, a nonhyphal species, is proposed for transfer to the mycelium forming genus Ambrosiozyma. Pichia toletana and Pichia xylosa form an independent lineage and are assigned to the genus Peterozyma, which is newly proposed.
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20. |
Khoury GA,
Fazelinia H,
Chin JW,
Pantazes RJ,
Cirino PC,
Maranas CD,
( 2009 ) Computational design of Candida boidinii xylose reductase for altered cofactor specificity. PMID : 19693930 : DOI : 10.1002/pro.227 PMC : PMC2786976 Abstract >>
In this study we introduce a computationally-driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non-native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR-GGD) being 27-fold more active on NADH. The NADPH-dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10(4)-fold change in specificity to NADH (CbXR-REG). The remaining two variants (CbXR-RTT and CBXR-EQR) had dual cofactor specificity for both nicotinamide cofactors.
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21. |
Osawa F,
Fujii T,
Nishida T,
Tada N,
Ohnishi T,
Kobayashi O,
Komeda T,
Yoshida S,
( 2009 ) Efficient production of L-lactic acid by Crabtree-negative yeast Candida boidinii. PMID : 19655300 : DOI : 10.1002/yea.1702 Abstract >>
Industrial production of L-lactic acid, which in polymerized form as poly-lactic acid is widely used as a biodegradable plastic, has been attracting world-wide attention. By genetic engineering we constructed a strain of the Crabtree-negative yeast Candida boidinii that efficiently produced a large amount of L-lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild-type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L-lactic acid by placing the bovine L-lactate dehydrogenase-encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar-fermenter resulted in an L-lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L-lactic acid-producing yeasts.
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22. |
Chin JW,
Khankal R,
Monroe CA,
Maranas CD,
Cirino PC,
( 2009 ) Analysis of NADPH supply during xylitol production by engineered Escherichia coli. PMID : 18698648 : DOI : 10.1002/bit.22060 Abstract >>
Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.
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23. |
Khankal R,
Chin JW,
Cirino PC,
( 2008 ) Role of xylose transporters in xylitol production from engineered Escherichia coli. PMID : 18359531 : DOI : 10.1016/j.jbiotec.2008.02.003 Abstract >>
Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.
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24. |
Sasano Y,
Yurimoto H,
Yanaka M,
Sakai Y,
( 2008 ) Trm1p, a Zn(II)2Cys6-type transcription factor, is a master regulator of methanol-specific gene activation in the methylotrophic yeast Candida boidinii. PMID : 18203863 : DOI : 10.1128/EC.00403-07 PMC : PMC2268522 Abstract >>
The methylotrophic yeasts are commonly used as hosts for heterologous gene expression. In this study, we describe a novel gene, TRM1, in Candida boidinii, responsible for the transcriptional activation of several methanol-inducible promoters. The encoded protein, Trm1p, is a Zn(II)2Cys6-type zinc cluster protein. Deletion of TRM1 completely inhibits growth on methanol but causes no growth defect on glucose or other nonfermentative carbon sources, glycerol, ethanol, or oleate. Trm1p is responsible for transcriptional activation of five methanol-inducible promoters tested, but not for peroxisome assembly or peroxisomal protein transport. Expression of the TRM1 gene was constitutive, and Trm1p localizes to the nuclei regardless of the carbon source. Two cis-acting methanol response elements (MREs), MRE1 and MRE2 are present in the promoter of the dihydroxyacetone synthase gene. Trm1p is shown to be required for MRE1-dependent methanol-inducible gene expression. Chromatin immunoprecipitation assays reveal that Trm1p binds to five methanol-inducible promoters upon methanol induction but does not bind in glucose-grown cells. Thus, the TRM1 gene encodes a master transcriptional regulator responsible for methanol-specific gene activation in the methylotrophic yeasts.
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25. |
Guo Q,
Gakhar L,
Wickersham K,
Francis K,
Vardi-Kilshtain A,
Major DT,
Cheatum CM,
Kohen A,
( 2016 ) Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii. PMID : 27100912 : DOI : 10.1021/acs.biochem.6b00181 PMC : PMC4917879 Abstract >>
The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 �� 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
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26. |
Garrard LJ,
Goodman JM,
( 1989 ) Two genes encode the major membrane-associated protein of methanol-induced peroxisomes from Candida boidinii. PMID : 2760051 : Abstract >>
A massive proliferation of peroxisomes occurs in the yeast Candida boidinii when methanol is utilized as the sole carbon source; these peroxisomes contain the enzymes which catalyze the initial steps of methanol utilization. The most abundant peroxisomal membrane-associated protein has an apparent molecular mass of 20 kDa and is termed PMP20. We report the isolation of two genes that encode very similar forms of PMP20; this is the first report of genes that encode proteins associated with peroxisomal membranes. Southern analysis demonstrates that the two genes are on different loci, although there are several homologous regions of both 5'- and 3'-untranslated sequence. One of the areas of 5' homology is within the untranslated region of the mRNA. Within the coding region there are 35 base differences between the two genes that are reflected in only five amino acid differences. The mRNAs representing both genes of PMP20 are induced in cells grown in methanol-containing medium and are below detection in cells grown in glucose. S1 nuclease protection analysis indicates that there is a 2.5-fold difference in mRNA expression between the two genes when induced. The predicted sequences of both PMP20 genes show the absence of a cleaved amino-terminal leader sequence and the presence of only 1 cysteine residue. In agreement with previous biochemical data suggesting a peripheral association of this protein with the membrane (Goodman, J. M., Maher, J., Silver, P. A., Pacifico, A., and Sanders, D. (1986) J. Biol. Chem. 261, 3464-3468), there are no obvious membrane spanning regions predicted in the sequences. Both PMP20 gene products contain the carboxyl-terminal sequence AKL, similar to the putative SKL peroxisomal sorting sequence (Gould, S. J., Keller, G.-A., and Subramani, S. (1988) J. Cell Biol. 107, 897-905).
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27. |
Zhai Z,
Yurimoto H,
( 2012 ) Molecular characterization of Candida boidinii MIG1 and its role in the regulation of methanol-inducible gene expression. PMID : 22711140 : DOI : 10.1002/yea.2909 Abstract >>
Methanol-inducible gene promoters in methanol-utilizing yeasts are used in high-level heterologous gene expression systems. Generally, expression of methanol-inducible genes is completely repressed by the presence of glucose. In this study we identified the MIG1 gene in Candida boidinii, which encodes a homologue of the glucose repressor Mig1p of Saccharomyces cerevisiae. Disruption of the CbMIG1 gene had no growth effect on various carbon sources. Activation of the methanol-inducible AOD1 gene, which encodes alcohol oxidase, was increased in the early stage of methanol induction when cells of the CbMIG1-disrupted strain were transferred from glucose medium to methanol medium. Furthermore, CbMig1p tagged with yellow fluorescent protein was primarily localized in the nucleus of glucose-grown cells, but was diffuse in the cytosol of methanol-grown cells. This cytosolic diffusion in methanol-grown cells occurred in a CbMsn5p-dependent manner. These results suggest that CbMig1p is involved in negative regulation of methanol-inducible gene expression in C. boidinii.
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28. |
McCammon MT,
Dowds CA,
Orth K,
Moomaw CR,
Slaughter CA,
Goodman JM,
( 1990 ) Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae. PMID : 2243083 : Abstract >>
A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.
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29. |
Zhai Z,
Yurimoto H,
Sakai Y,
( 2012 ) Msn5p is involved in formaldehyde resistance but not in oxidative stress response in the methylotrophic yeast Candida boidinii. PMID : 22313768 : DOI : 10.1271/bbb.110679 Abstract >>
Methylotrophic yeasts, which can utilize methanol as sole carbon and energy source, are exposed to two toxic metabolic intermediates, formaldehyde and hydrogen peroxide, during growth on methanol. Here we report that Msn5p, an importin-�] family nuclear exporter, participated in the formaldehyde resistance mechanism but not in the hydrogen peroxide resistance mechanism in Candida boidinii. Disruption of the MSN5 gene in this yeast caused retardation of growth on formaldehyde-generating growth substrates such as methanol and methylamine, but the expression levels of the methanol-metabolizing enzymes did not fall. The Msn5p-depleted strain was sensitive to formaldehyde but not to hydrogen peroxide. Furthermore, a yellow fluorescent protein-tagged Msn5p was diffuse in the cytoplasm of C. boidinii when the cells were treated with high concentrations of formaldehyde or ethanol, but was predominantly associated with the nuclei following treatment with hydrogen peroxide.
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30. |
( 1997 ) Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline. PMID : 9226256 : DOI : 10.1128/jb.179.14.4480-4485.1997 PMC : PMC179282 Abstract >>
The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.
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31. |
( 1993 ) PMP47, a peroxisomal homologue of mitochondrial solute carrier proteins. PMID : 8291088 : Abstract >>
N/A
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32. |
( 1996 ) The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme. PMID : 8698821 : DOI : 10.1083/jcb.134.1.37 PMC : PMC2120916 Abstract >>
Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.
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33. |
Sakai Y,
Marshall PA,
Saiganji A,
Takabe K,
Saiki H,
Kato N,
Goodman JM,
( 1995 ) The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae. PMID : 7592467 : DOI : 10.1128/jb.177.23.6773-6781.1995 PMC : PMC177542 Abstract >>
The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization.
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34. |
Moreno M,
Lark R,
Campbell KL,
Goodman JM,
( 1994 ) The peroxisomal membrane proteins of Candida boidinii: gene isolation and expression. PMID : 7871884 : DOI : 10.1002/yea.320101108 Abstract >>
Candida boidinii is a methylotrophic yeast in which several growth substrates can cause vigorous peroxisomal proliferation. While such diverse substrates as methanol, oleic acid and D-alanine induce different peroxisomal metabolic pathways, membranes seem to contain common abundant peroxisomal membrane proteins (PMPs). These proteins have been termed PMP31, PMP32 and PMP47. The gene encoding PMP47 has been previously cloned and analysed. We now report the isolation of a second PMP47 gene (or allele) as well as PMP31 and PMP32. PMP47A and PMP47B share 95% sequence identity at the amino acid level. PMP31 and PMP32 each contain 256 amino acids and are highly similar (97% identity) in protein sequence. Both PMP31 and PMP32 are predicted to span the membrane once or twice. All abundant PMPs of C. boidinii are basic in charge; they all have predicted isoelectric points above 10. RNAs corresponding to the PMP47s and to PMPs31-32 are strongly induced by methanol, oleic acid and D-alanine. While the PMP47s probably encode substrate carriers, the functions of PMP31 and PMP32 from C. boidinii are still unknown.
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35. |
Allen SJ,
Holbrook JJ,
( 1995 ) Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica. PMID : 7557425 : DOI : 10.1016/0378-1119(95)00347-9 Abstract >>
NAD-dependent formate dehydrogenase (FDH) was isolated from Candida methylica (Cm) grown on 0.5% methanol. Its N-terminal amino acid (aa) sequence was determined, as was that of a commercial FDH from Candida boidinii. Degenerate oligodeoxyribonucleotides were made to the 5' region of the fdh gene from Cm using this information and to the 3' region using C-terminal aa sequence data from the methylotropic yeast, Hansenula polymorpha. An almost complete 1.1-kb fragment was amplified from Cm genomic DNA via the polymerase chain reaction (PCR). This fragment was cloned, sequenced and used to probe a Southern blot, from which a 3.4-kb EcoRI fragment containing the fdh open reading frame (ORF) was isolated. The complete nucleotide sequence of this fdh ORF was determined and corresponds to a protein of 364 aa (40,343 Da). The ORF of fdh was cloned into pKK223-3 using PCR and transformed into Escherichia coli. Enzymatically active FDH was produced to 15% of soluble E. coli protein. The deduced aa sequence of this FDH is compared to the aa sequences of four known FDH, from bacteria, yeast, fungi and plant mitochondria.
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36. |
Shiraishi K,
Hioki T,
Habata A,
Yurimoto H,
Sakai Y,
( 2018 ) Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress. PMID : 29183915 : DOI : 10.1242/jcs.209114 Abstract >>
The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii, we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a �]-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress.This article has an associated First Person interview with the first author of the paper.
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37. |
( 2013 ) Relationships among genera of the Saccharomycotina (Ascomycota) from multigene phylogenetic analysis of type species. PMID : 22978764 : DOI : 10.1111/1567-1364.12006 Abstract >>
Relationships among ascomycetous yeast genera (subphylum Saccharomycotina, phylum Ascomycota) have been uncertain. In the present study, type species of 70 currently recognized genera are compared from divergence in the nearly entire nuclear gene sequences for large subunit rRNA, small subunit (SSU) rRNA, translation elongation factor-1�\, and RNA polymerase II, subunits 1 (RPB1) and 2 (RPB2). The analysis substantiates earlier proposals that all known ascomycetous yeast genera now assigned to the Saccharomycotina represent a single clade. Maximum likelihood analysis resolved the taxa into eight large multigenus clades and four-one- and two-genus clades. Maximum parsimony and neighbor-joining analyses gave similar results. Genera of the family Saccharomycetaceae remain as one large clade as previously demonstrated, to which the genus Cyniclomyces is now assigned. Pichia, Saturnispora, Kregervanrija, Dekkera, Ogataea and Ambrosiozyma are members of a single large clade, which is separate from the clade that includes Barnettozyma, Cyberlindnera, Phaffomyces, Starmera and Wickerhamomyces. Other clades include Kodamaea, Metschnikowia, Debaryomyces, Cephaloascus and related genera, which are separate from the clade that includes Zygoascus, Trichomonascus, Yarrowia and others. This study once again demonstrates that there is limited congruence between a system of classification based on phenotype and a system determined from DNA sequences.
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38. |
( 1998 ) Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii. PMID : 9811645 : PMC : PMC107661 Abstract >>
The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Delta strain), and the growth of the das1Delta strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Delta strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480-4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Delta strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.
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39. |
( 1997 ) Allergens of Aspergillus fumigatus and Candida boidinii share IgE-binding epitopes. PMID : 9412580 : DOI : 10.1164/ajrccm.156.6.9702087 Abstract >>
From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.
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